The vast majority of HIV infected individuals develop antibodies to the virus. In most cases the antibodies only target the autologous strain, but some individuals develop neutralizing serologic responses to a broad range of different viral isolates. These responses are of interest because passive transfer of monoclonal antibodies with broad neutralizing activity to humanized mice or monkeys prevents infection. On the basis of these observations it has been proposed that a vaccine that elicits broadly neutralizing antibodies would be protective against HIV. However, little is known about the nature ofthe broadly neutralizing response. Only a small number of patients have been studied to date, and the majority of these patients were selected because their serologic activity was focused on the CD4 binding site ofthe viral envelope spike. The long- term goals of this proposal are to characterize new broadly neutralizing antibodies in terms of their functions in vivo and to understand how HIV-1 develops resistance to these antibodies in vivo. To accomplish these goal we propose three specific aims. First, we will develop an in vivo assay to assess the ability of broadly antibodies to prevent viral entry in mice. HIV neutralization is currently assayed in vitro using TZM-bl cells. The new mouse model will be used to examine the relative efficacy of different monoclonal antibodies and the contribution of innate effector mechanisms to blocking HIV entry in vivo. Second, we will define the basis for development of resistance to broadly neutralizing antibodies in HIV infected humanized mice. We will use the information for structure based rational design approaches to iteratively enhance antibody breadth and potency. The ultimate goal of this part of the proposal is to determine which of the large group of currently available antibodies might be most useful for passive vaccine and immunogen design approaches. Finally, we will evaluate potential immunogens designed using structural and other data in knock-in mice containing human germline antibody precursor genes. Taken together, these experiments should help inform future clinical studies in which neutralizing antibodies might be considered for use in passive therapy or prevention studies and which of their targets would be most useful for immunization strategies.